Computational Study of Phosphate Vibrations as Reporters of DNA

Sep 23, 2015 - A combined experimental and computational study on the interaction of nitrogen mustards with DNA. Mahyar Bonsaii , Khodayar Gholivand ,...
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Computational Study of Phosphate Vibrations as Reporters of DNA Hydration D. J. Floisand and S. A. Corcelli* Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States ABSTRACT: The sensitivity of the phosphate asymmetric stretch vibrational frequency to DNA hydration was investigated with molecular dynamics (MD) simulations and a spectroscopic map relating the vibrational frequency to the electrostatics of its environment. 95% of the phosphate vibrational frequency shift in fully hydrated DNA was due to water within two hydration layers. The phosphate vibration was relatively insensitive to water in the major and minor grooves and to the sodium counterions but was enormously sensitive to water interacting with the DNA backbone. Comparisons to experimental measurements on DNA as a function of relative humidity suggest that one water molecule per phosphate group likely persists at the lowest values of the relative humidity. Finally, the calculated spectral diffusion dynamics show that water in the vicinity of the DNA backbone is slowed by a factor of ∼5, in agreement with NMR and solvation dynamics experiments, as well as previous MD simulations.

humidity of DNA in a range from ∼0 to 92%.5 In this range of relative humidity, the experiments found that the asymmetric stretch band of (PO2)− exhibits a continuous shift to lower frequencies of approximately −18 cm−1 with increasing hydration. Experiments on dioleoylphosphatidylcholine (DOPC) and dioleoylphosphoethanolamine (DOPE) reverse micelles showed a similar magnitude in the red shift of the phosphate asymmetric stretch band as the degree of hydration was controllably varied by changing the amount of water encapsulated within the reverse micelle.6 In contrast, the symmetric stretch of (PO2)− does not exhibit nearly as dramatic a shift in either the reverse micelle or DNA experiments. Motivated by the sensitivity of phosphate vibrations to hydration, Siebert et al. recently reported the first twodimensional infrared (2D IR) spectra of DNA in the 900− 1300 cm−1 regime.20 Their measurements were performed on DNA at 92% relative humidity, which should contain about two hydration layers and more than 20 water molecules per base pair.7 The 2D IR spectra for both the symmetric and asymmetric stretch were dramatically elongated along the diagonal, indicating the predominance of inhomogeneous broadening. From the 2D IR measurements, the frequency time correlation function (FTCF) was extracted. The FTCF encodes dynamical information on the environment in the vicinity of the phosphate group, insomuch as the environment causes the phosphate vibrational frequencies to fluctuate. In their analysis of the 2D IR line shapes, Siebert et al. assumed a biexponential form for the FTCF. The fast time constant was 300 fs, but the slower time constant could not be unambiguously determined because the vibrational population

W

ater is essential for the proper function of biological molecules, such as DNA, proteins, and phospholipids.1,2 The presence of water plays an integral role in biomolecular structural organization and is central to the assembly and threedimensional shape of proteins and nucleic acids. However, despite extensive experimental and simulation efforts,3−20 how the properties of water are altered in the vicinity of biomolecules is not yet fully understood. Vibrational spectroscopy of suitable infrared (IR) reporters offers the possibility of revealing the structural and dynamical properties of water at biomolecular interfaces with tremendous spatial specificity.21−23 This is because the vibrational frequencies of IR reporters are exquisitely sensitive to their local environment. Shifts in IR absorption spectra can often be related to changes in the structural properties of the solvent, and the time evolution of the vibrational frequencya process referred to as spectral diffusion, which is experimentally accessible in two-dimensional infrared (2D IR) spectroscopic measurementsreveals the dynamics of the environment in the immediate vicinity of the IR probe. From the experiments alone, however, it is difficult to extract specific molecular-level information about solvent structure and dynamics. Molecular dynamics (MD) simulations and models that relate the vibrational frequency to its instantaneous solvent environment greatly enhance the interpretation of vibrational spectroscopic measurements. Phosphate groups are ubiquitous in biology. They are found in a broad range of biomolecules, including phospholipid membranes, DNA, RNA, ADP, ATP, and phosphorylated proteins. The (PO2)− moiety contains both a symmetric and an antisymmetric stretch vibration that absorb at about 1100 and 1250 cm−1, respectively, and there have been a series of experimental studies investigating the IR spectroscopy of phosphate groups in biological contexts.6,18,20,24−30 Elsaesser and co-workers have measured the shift in the asymmetric phosphate IR absorption band as a function of the relative © 2015 American Chemical Society

Received: September 7, 2015 Accepted: September 23, 2015 Published: September 23, 2015 4012

DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017

Letter

The Journal of Physical Chemistry Letters

between any DNA atom and the closest box edge. Sodium counterions were added to establish charge neutrality, and all covalent bonds containing hydrogen were fixed at equilibrium lengths using the SHAKE algorithm.37 The resulting 30599 atom system, consisting of the DNA, 22 Na+ counterions, and 9939 water molecules, was energy minimized to relieve any residual unfavorable steric interactions introduced during the solvation procedure. First, the dodecamer was restrained (500 kcal/mol·Å2) while the water and counterions were subjected to 1000 steps of minimization. Then, the full system was allowed to relax during an additional 2500 steps of unrestrained minimization. The system was simulated for 150 ps in the NPT ensemble36 to establish a constant pressure of 1 atm and a constant temperature of 300 K. The simulation box was then isotropically scaled to the average volume of the last 50 ps of the NPT simulation. Equilibration proceeded for another 150 ps in the NVT ensemble,38,39 and the final velocities were scaled to represent a temperature of 300 K. A final equilibration of 600 ps was performed in the NVE ensemble. A 25 ns trajectory was produced in the NVE ensemble with configurations collected every 4 fs for analysis, which resulted in 6.25 million snapshots. In all of the MD simulations, a 2 fs time step was employed and the particle-mesh Ewald (PME)40 summation method was used to compute electrostatic interactions with a 9 Å real-space cutoff. The nonbonded interactions were also truncated at 9 Å. The spectroscopic map in eq 1 was utilized to compute shifts in the asymmetric stretch vibrational frequencies of the phosphate groups along the DNA backbone. In order to apply eq 1, the electric field due to the water and Na+ counterions was computed at the midpoint between the two oxygen atoms of each (PO2)− group and projected along the C2 symmetry axis, defined as the unit vector between the phosphate atom and the oxygen−oxygen midpoint, r̂C2. To appropriately capture the long-ranged contributions to the electric field, we used the damped shifted force (DSF) approach, in which the electric field due to a collection of N atomic-centered point charges, {qi}, is given in atomic units by

lifetime of the asymmetric (340 fs) and symmetric (1.24 ps) phosphate stretches are too short to capture the full extent of spectral diffusion. The 340 ps population lifetime is ubiquitous (i.e., the lifetime is similar for phosphates in different contexts). Therefore, it is likely that the energy redistribution pathway mostly involves the phosphate moiety itself. The slower time constant for the FTCF was greater than 10 ps. The fast time scale was assigned to fluctuations of the relatively static environment in the immediate vicinity of the phospholipid group, and the slow time scale was assigned to water− phosphate hydrogen bonds that persist beyond 10 ps. The combination of MD simulations and empirical spectroscopic maps that relate the vibrational frequencies of an infrared reporter to its local environment is a powerful approach for the interpretation of vibrational spectroscopic measurements in the condensed phase and in biological systems.31 Using density functional theory (DFT) calculations, Levinson et al. investigated the sensitivity to hydration of the phosphate vibrations in the model compound dimethyl phosphate (DMP).24 In the gas phase, a single water molecule was found to hydrogen bond to DMP in two distinctly different configurations that differ in energy by just 0.54 kcal mol−1. In the lower-energy configuration, the asymmetric phosphate stretch was blue-shifted by 9.3 cm−1, whereas a red shift of −34.8 cm−1 was observed for the higher-energy configuration. The authors found that these shifts in the asymmetric phosphate stretch vibrational frequency, δω, could be nearly quantitatively reproduced with a simple linear relationship δω = ω − ωg = cE

(1)

where ωg is the vibrational frequency in the absence of solvent and c is a parameter that was determined by applying a uniform electric field of magnitude E along the symmetry axis of the (PO2)− group of DMP in the gas phase and computing the shift in the phosphate asymmetric stretch vibrational frequency with DFT. In essence, c = 0.53 cm−1/(MV/cm) is the theoretically calculated Stark tuning rate of the phosphate asymmetric stretch. In the presence of explicit aqueous solvent, E is the projection of the electric field along the (PO2)− C2 symmetry axis calculated at the midpoint between the two oxygen atoms of the (PO2)− group. Henceforth, eq 1 will be referred to as a “spectroscopic map” because it relates the spectroscopic quantity of interest, ω, to other properties of the environment. In this case, eq 1 relates ω to the electric field of the surroundings. The water molecules, as in many conventional water force fields, were modeled as a collection of three atomiccentered point charges. Equation 1 then predicts a −29.3 cm−1 red shift for one of the DMP/water complexes and a 4.6 cm−1 blue shift for the other, which are in good agreement with the full DFT calculations of the change in vibrational frequencies, − 34.8 and 9.3 cm−1, respectively. In this Letter, we plan to utilize eq 1 in conjunction with MD simulations of DNA in explicit aqueous solution to establish a molecular-level understanding of how the asymmetric phosphate stretch vibration reports on DNA hydration. The fully hydrated MD simulations were constructed and rigorously equilibrated using a protocol previously reported.10,32,33 Briefly, all simulations were performed using AMBER 9.034 with the parm99 force field35 for DNA and the SPC/E water model.36 A high-resolution X-ray crystal structure of the extended A-tract DNA dodecamer d(CGCAAATTTGCG) (Protein Data Bank code 1S2R), including crystallographic water molecules, was solvated in a cubic periodic box with a minimum buffer of 10 Å

⎯⇀ ⎯

E DSF =

⎧ ⎡⎛ erfc(ar ) ⎪ j

∑ ⎨qi⎢⎜⎜ i

⎩ ⎢⎣⎝ ⎪

ri 2

+

2 2 2α exp(−α ri ) ⎞ ⎟⎟ ri π 1/2 ⎠

2 2 ⎤⎫ ⎛ erfc(αR ) 2α exp(−α R c ) ⎞⎥⎪ c ⎟ ⎬r ̂ −⎜ + 2 ⎪ Rc π 1/2 ⎝ Rc ⎠⎥⎦⎭

ri ≤ R c (2)

The DSF method is a pairwise alternative to the Ewald summation technique, which is comparably accurate.41 In eq 2, ri is the distance between charge qi and the point at which the electric field is being computed, r̂i is the unit vector directing from the midpoint between the phosphate oxygen atoms and the charge qi, and Rc is a spherical cutoff radius. Only charges with distances less than Rc = 12 Å contribute to the sum in eq 2. α = 0.2 Å−1 is a damping parameter whose value was optimized by Fennel and Gezelter.41 The magnitude of the electric field needed to apply eq 1 was E = Ê ·r̂C2. The calculated average shift in the asymmetric phosphate stretch in fully hydrated DNA was −41.4 cm−1. Very little variation was observed in the magnitude of the frequency shift along the DNA strand (standard deviation = 0.3 cm−1). It should be noted that the magnitude of the frequency shift is approximately double of what is observed in the experiments 4013

DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017

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The Journal of Physical Chemistry Letters by Elsaesser and co-workers in a relative humidity range from ∼0 to 92%. The source of this discrepancy will be discussed extensively below as we seek to understand the detailed molecular-level factors that contribute to the frequency shift. One of the advantages of the electrostatics-based spectroscopic map in eq 1 is the ease of determining the contributions of individual water molecules and sodium counterions to the phosphate vibrational frequency shift. This is accomplished by simply restricting which point charges are included in the sum in eq 2. For example, by including only the sodium counterions in the sum, their average contribution of just −2.1 cm−1 to the frequency shift was determined. It should be noted that changing the cutoff radius in eq 2 does not affect this result; the electric field is fully converged within 12 Å. Thus, we can immediately conclude that the sodium counterions are not having a substantial direct effect, on average, compared to water. Consistent with the earlier DNA simulation studies of Young, Ravishanker, and Beveridge, about 80% of the counterions are (cylindrically) located within 12 Å of the DNA backbone.42 However, because the ions influence both the hydration structure and contribute to the stability of DNA, they still have an indirect influence on the phosphate vibrational frequencies. In Figure 1, the average contribution

Figure 2. (top) Average shift of the phosphate asymmetric stretch vibrational frequency in double-stranded DNA from the closest N = 1−6 water molecules. (bottom) Representative image of a phosphate group with its six closest hydration water molecules.

however, gives a small blue shift of 0.4 cm−1, and the sixth water molecule results in a another 1.0 cm−1 blue shift. Additional water molecules (not shown) cause the frequency to monotonically shift to the red until convergence is achieved. A representative image showing a phosphate group along with its closest six water molecules is shown in the bottom panel of Figure 2. The locations of the water molecules are in excellent agreement with crystallographic measurements on doublestranded DNA.43 The closest four water molecules are directly hydrogen bonding to the oxygen atoms of the (PO2)− group and collectively contribute −34.6 cm−1 to the asymmetric phosphate stretch vibrational frequency. The fifth and sixth closest water molecules are not primarily hydrogen bonding with the oxygen atoms of the (PO2)− group but are instead interacting with the oxygen atoms to which the (PO2)− group is covalently linked. These water molecules collectively give a 9.0 cm−1 blue shift. This observation also agrees with previous DFT investigations of DMP complexed with one water molecule in the gas phase.24 There, two different configurations of the water moleculeone in which the water molecule hydrogen bonds directly to the (PO2)− group and another where it interacts with the oxygen atoms to which the (PO2)− is linkedresulted in phosphate asymmetric stretch frequency shifts of −34.8 and 9.3 cm−1, respectively. Another perspective on the sensitivity of phosphate vibrations in DNA comes from investigating the effects of different zones of hydration. First, we calculated the effect of water in the first and second hydration shells of DNA on the phosphate asymmetric stretch vibrational frequency. The cutoff for the first hydration shell was defined as R1 = f(rw + rs), where rw and rs are the van der Waals radii of the water oxygen atom and that of the nearest DNA atom, respectively; the coefficient f was chosen as 1.1, a value used previously in hydration studies

Figure 1. Average shift of the phosphate asymmetric stretch vibrational frequency in double-stranded DNA from water and sodium counterions within a specified spherical distance of the phosphate group, defined with respect to the midpoint between the phosphate oxygen atoms.

of water and ions within a specified spherical distance of the phosphate group, defined with respect to the midpoint between the phosphate oxygen atoms, to the shift in the phosphate asymmetric stretch vibrational frequency is shown. The change in frequency is significantly nonmonotonic. There is a continuous red shift to −34.9 cm−1 at 3.75 Å, followed by a modest blue shift of 2.6 cm−1 over the course of the next 0.75 Å, and finally another red shift until the frequency is converged to −40.7 cm−1 within 12 Å. Qualitatively, the frequency shift is dominated by the solvent environment within approximately two hydration layers of the phosphate group, but the origin of the nonmonotonic behavior requires additional analysis. In the top panel of Figure 2, the average shift in the phosphate asymmetric stretch frequency due to the closest N water molecules is shown for N = 1−6. Interestingly, the calculated shift was, once again, nonmonotonic. On average, the closest water molecule leads to a −19.1 cm−1 red shift in the vibrational frequency. Including the four closest water molecules in the frequency calculation results in a continuous red shift to −34.9 cm−1. Adding a fifth water molecule, 4014

DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017

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The Journal of Physical Chemistry Letters of proteins8 and DNA.32 Water residing within a second cutoff radius, R2 = f(3rw + rs), representing R1 plus the diameter of an additional water molecule, was considered to be in the second DNA hydration shell. The results of the analysis are shown in Table 1. Water in the first hydration shell accounts for a −28.2

theory and experiment is reconciled if we assume that DNA is hydrated by one water molecule per phosphate group at low relative humidity. Finally, the MD simulations and the associated frequency analysis using the spectroscopic map in eq 1 allow for calculations of the spectral diffusion dynamics of the phosphate asymmetric stretch in fully hydrated DNA. The spectral diffusion dynamics are characterized by the normalized FTCF

Table 1. Average Phosphate Asymmetric Stretch Vibrational Frequency Shifts for the First and Second Hydration Shells of DNAa hydration zone first shell

second shell full hydration

major groove minor groove backbone total

C(t ) =

frequency shift (cm−1) 0.6 1.3 −30.1 −28.2 −11.0 −41.4

⟨δω(0)δω(t )⟩ C(0)

(3)

where δω(t) = ω(t) − ⟨ω⟩ is the fluctuation of the instantaneous vibrational frequency from its average value. C(t) is shown as an average over all phosphate groups in the simulation in Figure 3. It should be noted that the FTCF does

The first hydration shell is further decomposed into contributions from water in the major groove, minor groove, and the backbone of DNA.

a

cm−1 red shift. Water in the second solvation shell gives another −11.0 cm−1 red shift. Combined together, water in the first two hydration shells represent 95% of the full hydration shift. The water in the first hydration shell can be further decomposed into three spatial zones: major groove, minor groove, and backbone. Water associated directly with the DNA backbone has, by far, the largest influence on the phosphate asymmetric stretch vibrational frequency, resulting in a −30.1 cm−1 red shift. Interestingly, water in the major and minor groove result in small blue shifts of 0.6 and 1.3 cm−1, respectively. This is consistent with the blue shifts observed for the fifth and sixth closest water molecules to the phosphate group, which are typically located in the major and minor grooves. As noted previously, there is a disagreement between the calculated average shift in the phosphate asymmetric stretch vibrational frequency in fully hydrated DNA, −41.4 cm−1, and the experimental shift of −18 cm−1 at 92% relative humidy.5 There are several possible reasons for the discrepancy. One possibility is that the frequency map, developed for DMP in uniform electric fields and validated for a pair of DMP·H2O complexes, is not fully transferable to phosphate vibrations in hydrated DNA. Although the detailed quantitative accuracy of the simple spectroscopic map in eq 1 merits further study, there is another interesting hypothesis for the discrepancy; the ∼0% relative humidity experiments are not completely dry. There is experimental evidence that supports this explanation. Gravimetric measurements at low relative humidity suggest the presence of up to one water molecule per phosphate group of residual hydration.7 If the residual water is indeed interacting with the charged phosphate group, it would cause a substantial red shift in the phosphate asymmetric stretch vibrational frequency. Our calculations predict that the shift would be −19.1 cm−1. The same gravimetric experiments observe six water molecules per phosphate group at 75% relative humidity. The experimentally measured frequency shift is −15 cm−1 at 75% relative humidity, and the calculated frequency difference between one and six water molecules is −14.5 cm−1. At 92% relative humidity, the gravimetric experiments suggest there are two hydration layers around DNA, corresponding to an experimental frequency shift of −18 cm−1. The difference between the two hydration layer frequency shift and the one water frequency shift is −19.1 cm−1. The disagreement between

Figure 3. (black) Calculated frequency fluctuation time correlation function, C(t), for the phosphate asymmetric stretch vibration in fully hydrated DNA. (red) A triexponential fit of C(t).

not vary significantly for individual phosphate groups, even for those near the ends of the double-stranded dodecamer. Also shown in Figure 3 is a triexponential fit of the calculated FTCF. The time constants of the fit are shown in Table 2. The FTCF Table 2. Time Constants for the Tri-Exponential Fit of the Calculated Frequency Fluctuation Time Correlation Function in Figure 3 along with Experimental Time Constants Extracted from 2D IR Measurements on DNA at 92% Relative Humidity, Shown for Comparison 20 theory theory (weighted average of τ1 and τ2) experiment20

τ1 (ps)

τ2 (ps)

τ3 (ps)

0.052 0.31 0.3

1.2

7.1 7.1 >10

is primarily reporting on the dynamics of water because the solvent motion is what causes the phosphate frequency to fluctuate. Moreover, because we have established that the phosphate frequencies are most sensitive to hydration within two solvent shells, the FTCF is encoding information about the dynamics of water in the immediate vicinity of DNA. Of particular interest is the slowest time constant, 7.1 ps. From 2D IR measurements, bulk water is known to rearrange on a 1.5 ps time scale;44 thus, 7.1 ps represents a significant slowing of water dynamics by almost a factor of 5. This is in excellent agreement with NMR experiments,17 solvation dynamics experiments, 1 3 and previous MD simulations of DNA.10,16,19,32,33 Siebert et al. recently reported 2D IR 4015

DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017

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The Journal of Physical Chemistry Letters

(5) Szyc, Ł.; Yang, M.; Nibbering, E. T. J.; Elsaesser, T. Ultrafast Vibrational Dynamics and Local Interactions of Hydrated DNA. Angew. Chem., Int. Ed. 2010, 49, 3598−3610. (6) Costard, R.; Heisler, I. A.; Elsaesser, T. Structural Dynamics of Hydrated Phospholipid Surfaces Probed by Ultrafast 2D Spectroscopy of Phosphate Vibrations. J. Phys. Chem. Lett. 2014, 5, 506−511. (7) Falk, M.; Hartman, K. A.; Lord, R. C. Hydration of Deoxyribonucleic Acid I. Gravemtric Study. J. Am. Chem. Soc. 1962, 84, 3843−3846. (8) Pizzitutti, F.; Marchi, M.; Sterpone, F.; Rossky, P. J. How Protein Surfaces Induce Anomalous Dynamics of Hydration Water. J. Phys. Chem. B 2007, 111, 7584−7590. (9) Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.; Gruebele, M.; Leitner, D. M.; Havenith, M. An Extended Dynamical Hydration Shell around Proteins. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 20749−20752. (10) Furse, K. E.; Corcelli, S. A. Molecular Dynamics Simulations of DNA Solvation Dynamics. J. Phys. Chem. Lett. 2010, 1, 1813−1820. (11) Abbyad, P.; Shi, X.; Childs, W.; Mcananey, T. B.; Cohen, B. E.; Boxer, S. G. Measurement of Solvation Responses at Multiple Sites in a Globular Protein Measurement of Solvation Responses at Multiple Sites in a Globular Protein. J. Phys. Chem. B 2007, 111, 8269−8276. (12) Pal, S. K.; Zewail, A. H. Dynamics of Water in Biological Recognition. Chem. Rev. 2004, 104, 2099−2123. (13) Pal, S. K.; Zhao, L.; Zewail, A. H. Water at DNA Surfaces: Ultrafast Dynamics in Minor Groove Recognition. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 8113−8118. (14) Golosov, A. A.; Karplus, M. Probing Polar Solvation Dynamics in Proteins: A Molecular Dynamics Simulation Analysis. J. Phys. Chem. B 2007, 111, 1482−1490. (15) Halle, B.; Nilsson, L. Does the Dynamic Stokes Shift Report on Slow Protein Hydration Dynamics? J. Phys. Chem. B 2009, 113, 8210− 8213. (16) Furse, K. E.; Corcelli, S. A. The Dynamics of Water at DNA Interfaces: Computational Studies of Hoechst 33258 Bound to DNA. J. Am. Chem. Soc. 2008, 130, 13103−13109. (17) Halle, B. Protein Hydration Dynamics in Solution: A Critical Survey. Philos. Trans. R. Soc., B 2004, 359, 1207−1223. (18) Fogarty, A. C.; Laage, D. Water Dynamics in Protein Hydration Shells: The Molecular Origins of the Dynamical Perturbation. J. Phys. Chem. B 2014, 118, 7715−7729. (19) Fogarty, A. C.; Duboué-Dijon, E.; Sterpone, F.; Hynes, J. T.; Laage, D. Biomolecular Hydration Dynamics: A Jump Model Perspective. Chem. Soc. Rev. 2013, 42, 5672−5683. (20) Siebert, T.; Guchhait, B.; Liu, Y.; Costard, R.; Elsaesser, T. Anharmonic Backbone Vibrations in Ultrafast Processes at the DNA− Water Interface. J. Phys. Chem. B 2015, 119, 9670−9677. (21) Lindquist, B. A.; Furse, K. E.; Corcelli, S. A. Nitrile Groups as Vibrational Probes of Biomolecular Structure and Dynamics: An Overview. Phys. Chem. Chem. Phys. 2009, 11, 8119−8132. (22) Getahun, Z.; Huang, C. Y.; Wang, T.; Leon, B.; DeGrado, W. F.; Gai, F. Using Nitrile-Derivated Amino Acids as Infrared Probes of Local Environment. J. Am. Chem. Soc. 2003, 125, 405−411. (23) King, J. T.; Kubarych, K. J. Site-Specific Coupling of Hydration Water and Protein Flexibility Studied in Solution with Ultrafast 2D-IR Spectroscopy. J. Am. Chem. Soc. 2012, 134, 18705−18712. (24) Levinson, N. M.; Bolte, E. E.; Miller, C. S.; Corcelli, S. A.; Boxer, S. G. Phosphate Vibrations Probe Local Electric Fields and Hydration in Biomolecules. J. Am. Chem. Soc. 2011, 133, 13236−13239. (25) Chen, X.; Hua, W.; Huang, Z.; Allen, H. C. Interfacial Water Structure Associated with Phospholipid Membranes Studied by PhaseSensitive Vibrational Sum Frequency Generation Spectroscopy. J. Am. Chem. Soc. 2010, 132, 11336−11342. (26) Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. Three Distinct Water Structures at a Zwitterionic Lipid/Water Interface Revealed by Heterodyne-Detected Vibrational Sum Frequency Generation. J. Am. Chem. Soc. 2012, 134, 7842−7850. (27) Zhao, W.; Moilanen, D. E.; Fenn, E. E.; Fayer, M. D. Water at the Surfaces of Aligned Phospholipid Multibilayer Model Membranes

measurements on DNA at 92% relative humidity in the range from 900 to 1300 cm−1.20 The time constants of the FTCF for the asymmetric phosphate stretch were extracted from the 2D IR measurements using a biexponential Kubo ansatz. One time constant was 0.3 ps, and the other was not determined but was estimated as greater than 10 ps. Our calculation of 7.1 ps is clearly somewhat faster than the prediction of a slow time scale greater than 10 ps. However, the hydrogen bond rearrangement dynamics of the SPC/E water model are too fast by almost a factor of 2,44 which could certainly be a factor in the discrepancy. Interestingly, the amplitude weighted average of our two faster time scales, 0.31 ps, is in excellent agreement with experiment, 0.3 ps. Overall, the qualitative agreement with experiment is encouraging. Using MD simulations and a simple spectroscopic map, we have investigated the sensitivity of the phosphate asymmetric stretch in DNA to its hydration environment. Most importantly, the role of water interacting with the backbone of DNA was the most crucial determinant of the phosphate vibrational frequency and its time evolution. Several related molecular-level insights were gained: (1) the vibrational frequency is sensitive to water within two solvation shells of DNA and most sensitive to water molecules directly donating hydrogen bonds to the phosphate group; (2) water in the major and minor grooves of DNA has a negligible effect on the phosphate vibrational frequencies; (3) the sodium counterions also have a small average effect on the frequencies; (4) experiments on DNA as a function of relative humidity possibly contain one water molecule per phosphate group at the lowest values of the relative humidity; and (5) the spectral diffusion dynamics of the phosphate asymmetric stretch frequency report on the motion of water molecules interacting with the backbone of DNA and are slowed by a factor of ∼5. Vibrational spectroscopy of phosphate vibrations holds tremendous promise to report on local structure and dynamics in a variety of biophysical contexts (DNA, phosphorylated proteins, phospholipid membranes, etc.), and the interplay of experiment, theory, and simulation is essential to derive maximum physical insight.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Science Foundation (CHE-0845736). In addition, the authors are thankful for support from the Center for Research Computing at the University of Notre Dame and for helpful discussions with Dr. Kristina Davis.

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REFERENCES

(1) Chaplin, M. Do We Underestimate the Importance of Water in Cell Biology? Nat. Rev. Mol. Cell Biol. 2006, 7, 861−866. (2) Saenger, W.; Hunter, W. N.; Kennard, O. DNA Conformation is Determined by Economics in the Hydration of Phosphate Groups. Nature 1986, 324, 385−388. (3) Westhof, E. Water: An Integral Part of Nucleic Acid Structure. Annu. Rev. Biophys. Biophys. Chem. 1988, 17, 125−144. (4) Ball, P. Water as an Active Constituent in Cell Biology. Chem. Rev. 2008, 108, 74−108. 4016

DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017

Letter

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DOI: 10.1021/acs.jpclett.5b01973 J. Phys. Chem. Lett. 2015, 6, 4012−4017